Title: The molecular mechanisms of nutrient- and stress-dependent mTORC1 regulation mediated by human Sestrin2. Project Summary The mTOR complex 1 (mTORC1) is an important nutrient sensor whose chronic activation by overnutrition can provoke diverse metabolic pathologies such as insulin resistance and type II diabetes. Most pharmacological inhibitors of mTORC1, however, non-specifically suppress mTORC2?another mTOR complex that is critical for mediating insulin signal transduction?and thus inappropriate for diabetes treatment. Sestrins (Sesns) are recently identified mTORC1 suppressors. mTORC1-inhibitory function of Sesns attenuates development of most hypernutrition- and obesity- associated metabolic pathologies. Importantly, Sesns does not inhibit mTORC2 and rather upregulates its activity by suppressing mTORC1. Correspondingly, transgenic Sesn overexpression was highly effective in protecting liver from chronic mTORC1 activation, development of insulin resistance and progression of diabetic pathologies. These results suggest that Sesns and their downstream signaling pathway may have a therapeutic potential as a drug target toward the obesity-associated diseases. Recently, a number of studies by our labs and others have clarified the molecular targets of Sesns, which led to a clearer understanding of how Sesns inhibit mTORC1. As a result of extensive genetics and cell biology studies, a clear epistatic relationship between GATOR2, GATOR1, Rag GTPases and mTORC1 was established. Furthermore, a couple of recent papers also suggested that an amino acid leucine can bind to Sesns and modulates their activities. Despite its physiological significance, the biochemical and molecular basis by which these proteins interact with and signal to each other is still completely unknown. Without an understanding of the molecular level mechanism, it is nearly impossible to rationally design chemical probes to modulate this signaling cascade. As a part of this effort, we have recently determined the first crystal structure of human Sestrin2 (hSesn2), and are currently planning to use it as a starting platform for understanding the Sesn-dependent signal transduction pathway. Our long-term goal is to define the biochemical and structural properties of each signaling component within the Sesn-dependent signaling cascade and to reveal druggable structural motifs that are critical for functionality of this signaling pathway. Using a combination of X-ray crystallography, molecular electron microscopy (EM), cell biology and Drosophila/mouse genetics experiments, we will elucidate the structural, biochemical and cell-biological role of hSesn2 and its signaling intermediates?GATOR1 and GATOR2?in mTORC1 suppression. The molecular mechanisms and structural motifs identified from these studies are expected to reveal many new drug targets for future development of mTORC1-modulating pharmaceutical agents, which will be clinically significant for reducing the progression of diverse metabolic pathologies caused by hypernutrition and obesity.